Uncontrolled release of pollutants and untreated effluents into the environment, environmental pollution is one of the biggest issues faced globally. Environmental contamination is mostly caused by population increase, industrialization, exploration, urbanization, and mining.¹ The survival of humanity is seriously threatened by the significant amount of toxins that have been dumped into the environment, ranging from untreated sewage to nuclear waste.² Anthropogenic activities including industrialization, farming methods, population growth, and unhealthy competition for dominance are having severely negative effects on Earth. Pollutants, mostly phenols, polyaromatic hydrocarbons, pesticides, azo dyes, polychlorinated chemicals, heavy metals, and other hazardous substances, are produced as a result of these processes. These chemicals endanger the biotic components of ecosystems because they are resistant to biodegradation.³ These contaminants have a profound impact on every area of the planet, having carcinogenic, mutagenic, and toxic consequences on people and other living things.⁴

Clean water is essential for life, but it is often contaminated by industrial activities, leading to wastewater. This pollution, containing pesticides, pharmaceutical wastes, plastics, heavy metals, organic solvents, acids, alkali, biological wastes, and xenobiotic compounds, poses significant environmental and health risks. To address these issues, conventional methods like physical, chemical, and biological treatments are insufficient. Nanotechnology and nanofiltration are promising alternatives for industrial wastewater treatment due to their high surface area, eco-friendliness, and effectiveness in eliminating pollutants.⁵

Micropollutants (MPs) are a growing concern in wastewater due to human activities. Despite their low presence, MPs are highly toxic and non-degradable, causing global water quality and health hazards. Conventional wastewater treatment is inefficient and produces hazardous sludge. Alternative sustainable approaches are needed, and recent micro-biotechnological methods, such as bio-adsorption, membrane filtration, biocatalysts, bioreactors, and nanotechnology, have shown effectiveness in removing MPs. Bacteria, fungi, and microalgae can degrade MPs through biosorption, biotransformation, bioaccumulation, and bioconversion mechanisms. Microbial-based nanoparticles (MNPs) can be integrated into existing membrane or adsorption technologies, but systematic studies are still in its infancy.⁶ Micropollutants, including pesticides, pharmaceuticals, and personal care products, have improved human quality of life but are increasingly contaminating aqueous systems due to seepage, run-off, effluent discharge, and environmental uptake. These pollutants have significant ecotoxicities and adverse effects on human health, making them a critical environmental concern. Current remediation methods are costly, consume excess chemicals, require high energy, and create harmful by-products. However, oxidative laccases and peroxidases, microalgae, and microalgae-bacteria consortia are emerging as green alternatives for remediating micropollutants in contaminated systems. These methods can be used as secondary treatment techniques for wastewater plant effluents or as tertiary treatments in conjunction with other chemical and biological technologies.⁷ Pollutants have been cleaned up using a variety of physical and chemical techniques, including oxidising agents, electrochemical processes, pollutant adsorption, ion exchange, and membrane filtering. Traditional procedures were adequate for the high concentration of contaminants, but they were insufficient to reduce the pollution to allowable levels.⁸ Traditional methods for cleaning up pollutants have several drawbacks, including their high cost, difficult procedures, stringent international regulations imposed on decontamination, general public rejection, non-specificity, space limitations, and potential for secondary pollution creation.⁹ As a result, interest has grown in bioremediation, eco-friendly, and biological procedures. Plastic, introduced in the mid-20^th century, has been crucial for modern civilization due to its convenience and cost-effectiveness. However, its extensive use has led to significant environmental issues, including waste management issues in landfills and natural ecosystems.⁹ Microplastics, particularly those from polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC), have a profound negative impact on ecosystems and species health.¹⁰ Enzyme technology is crucial in promoting environmental conservation by converting plastic waste into less harmful compounds. Research on Thermobifida fusca hydrolase has led to the discovery of various enzymes that can break down PET, with PETase enzyme from Ideonella sakaiensis being particularly noteworthy for its ability to effectively degrade PET into intermediates like BHET, MHET, and TPA. MHETase further processes MHET to produce terephthalic acid and ethylene glycol.¹¹ Significant progress has been achieved in the domain of enzymatic microplastic degradation, specifically regarding polystyrene (PS). Advancements in this domain are apparent, as numerous microbes, including bacteria and fungi, possess the capability to decompose PS. Nonetheless, the specific enzymes that initiate this breakdown process are not yet well comprehended.¹²

Microorganisms that break down many contaminants enzymatically and convert them into less harmful compounds or metabolites that could be beneficial products are the mainstay of the bioremediation process.¹³ Reducing environmental pollution through the use of biological agents is an affordable process and reduces the risks they impose on people’s health and threats to the environment. To break down and detoxify pollutants, it mostly employs intracellular accumulation or enzymatic transformation.⁹ Microbial enzymes are regarded as innovative, economical, and promising when used in the bioremediation of persistent pollutants (Figure 1). Microbes are an abundant source of biocatalytic enzymes, essential in industries like textiles, agriculture, and pharmaceuticals. They reduce environmental pollution through biodegradation and bioremediation. The global enzyme market is growing due to their eco-friendliness, stability, and easy modification. Alternatives like recombinant DNA technology and protein engineering are used to create novel products.¹⁴ Because of their metabolic activity and their ability to thrive in various environmental conditions, bacteria, fungi, etc. are present throughout the biosphere and produce enzymes. Among the various microbial enzymes, such as those from Mycobacterium, Pseudomonas, Sphingomonas, Rhodococcus, Mycobacterium has already been shown to break down pesticides and hydrocarbons. Cytochrome P450s, dehydrogenases, laccases (Lac), proteases, hydrolases, lipases, and dehalogenases are the most prevalent microbial enzymes involved in bioremediation.¹⁵ The bioremediation technique has some constraints. Only a few biological agents have the aptness to produce specialized enzymes with sufficient power to break down contaminants, and the process is exceedingly slow and constant. Genetically engineered microbes produce required enzymes in huge quantities under ideal conditions; we choose them for bioremediation. Industrial microbial enzymes currently have a very competitive global market. Worldwide recognized companies in the enzyme production sector are DSM, Novozymes, Amano Enzymes Inc., and DuPont. By employing enzymes, we can safeguard our planet against the accumulation of hazardous and poisonous waste in the environment.¹⁶ Numerous novel microbial enzymes are been developed for various bioprocesses using rDNA technology, meta-genomics, and protein engineering. To increase the production and efficacy of various microbial enzymes, a range of molecular approaches are used in the food, paper, pharmaceutical, biotechnological, textile, leather, and other industries.^17,18 The major focus of this review are the function and mechanism of microbial enzymes in the sustainable bioremediation of industrial pollutants. Additionally, there is a gap in suitable approaches considering the bioremediation of toxic multi-pollutants using enzymatic applications. Most of the research remains lab-scale, with few studies on pilot or field applications. Enzyme stability and activity under real environmental conditions (e.g., temperature fluctuations, mixed contaminants, pH changes) are underexplored. The mechanisms of enzymatic degradation for complex pollutants (e.g., microplastics, PFAS, pharmaceutical residues) are not fully understood. The mechanisms of enzymatic degradation for complex pollutants (e.g., microplastics, PFAS, pharmaceutical residues) are not fully understood. Few sustainable, low-cost carriers (e.g., biochar, waste-derived supports) should have been optimized for enzyme immobilization. Focus is often on well-known microbes; the microbiomes of extreme environments (e.g., deep-sea vents, deserts, Antarctic soils) remain untapped for novel enzymes.

Production, optimization, and application of microbial enzymes for industrial pollution
Enzymes are efficient biocatalysts for various biochemical reactions, producing few by-products compared to chemical catalysts. Industrial enzymes, such as proteases, amylases, cellulases, and lipases, can be obtained from microorganisms through optimization and enzyme engineering. These enzymes are essential for industrial conditions, as wild microbial strains produce less enzyme than engineered microorganisms. Most industrial enzymes are derived from plants, animals, and microorganisms due to better yields, cost reduction, and reduced labor. Industrial enzymes are essential for various industries, used for commercial purposes (Figure 2). They are produced by microorganisms, which involve various steps such as isolation, screening, optimization of process parameters, fermentation, purification, characterization, formulation for sale, customer liaison, and working with regulatory authorities. Most bacteria and fungi used to produce industrial enzymes are genetically modified to overproduce them. Solid state and submerged fermentation are commonly used, but submerged fermentation is preferred due to the extracellular nature of the enzyme. Criteria such as pH and temperature stability, specificity, influence of activators and inhibitors, and reaction velocity are used in selecting industrial enzymes. Industrial enzymes are typically produced under controlled conditions using microorganisms, mainly bacteria and fungi being the main producers (Table 1 and 2). Industrial enzymes are frequently produced by submerged and solid-state fermentation. However, due to the extracellular character of the industrial enzyme that is released into the production medium, submerged fermentation has been consistently described as the preferred technique for industrial enzyme secretion from microorganisms. According to Sarrouh et al.,¹⁹ some of the factors taken into consideration when choosing which industrial enzymes to manufacture from microbes include pH and temperature stability, specificity, the impact of activators and inhibitors, and reaction velocity. Figure 2 demonstrates the comparative study between various microbial enzymes.

Table (1):
Bioremediation of industrial pollutants through various bacterial enzymes

Table (2):
Bioremediation of industrial pollutants through various fungal enzymes

Optimization of parameters for enzyme production by microorganisms
A number of factors, including incubation duration, agitation/shaking, pH, inoculum concentration, incubation temperature, carbon supply, metal ions, and nitrogen source, primarily affect the industrial enzymes produced by microorganisms. Improving the industrial enzyme yield is largely dependent on optimizing these parameters.²⁰

Application of microbial enzymes for the bioremediation of industrial pollutants
In the present scenario, microbial enzyme-mediated bioremediation is a viable approach for reduction and removal of hazardous industrial pollutants. Enzymes are present in almost every naturally existing organism i.e., from prokaryotes to eukaryotes.¹⁵ Many microbial enzymes act as catalysts for producing a variety of products from a variety of substrates under controlled circumstances. Additionally, through bioconversion or biodegradation processes, several microbial enzymes can quickly convert hazardous substances into valuable by-products (Figure 1).

Because they lower the reaction activation energy without having any long-term effects, enzymes speed up a variety of biological processes that are essential to maintaining human life. The production of enzymes from microorganisms offers a number of benefits, such as simple gene manipulation, quick growth under regulated conditions, ease of handling, increased manufacturing yield, etc.¹⁶

Microbial enzymes are also being employed more frequently in industrial settings as a result of their catalytic activity, non-toxicity, specificity, eco-friendliness, stability, cost-effectiveness, and ease of manufacturing.⁹ The sources, mechanisms involved, and uses of microbial enzymes isolated from different bacterial and fungal species for bioremediation are described in Table 1 and 2, respectively.

Cytochrome P450 (EC 1.14.14.1)
A member of the heme enzyme superfamily, cytochrome P450 is found extensively throughout the three domains of life-bacteria, archaea, and eukaryote.²¹ It carries out a variety of tasks, such as the biotransformation of harmful compounds in our ecosystem and synthesizing intricate natural products in living systems. They combine molecular oxygen with NADH or NADPH to produce oxidized products and a carbon substrate. They function as a cofactor.²² P450s can degrade xenobiotics naturally.²³ For catalytic activity, they also require ferredoxin and ferredoxin reductase as an electron source. Research on microbial P450s for the bioremediation of hydrocarbons and organic pollutants has been done utilizing both non-engineering and protein engineering approaches.²⁴

One of the recognized microbial P450s is the Bacillus megaterium CYP102A1 (P450BM3) model, which has been proven through protein engineering research to have the ability to oxidize PAHs.²² Many microorganisms, mainly bacteria such as Rhodococcus, Gordonia, Mycobacterium, Pseudomonas, etc., have been found to include some catabolic genes and plasmids that express P450s for the reduction of POPs (persistent organic pollutants) and their elimination from our environment.²⁵

Laccases (EC 1.10.3.2)
Laccases are extracellular enzymes composed of several glycoprotein subunits and contain multiple copper ions. Laccases oxidize certain phenolic and aromatic chemicals in bacteria, fungi, and plants, as well as certain amines, ethers, and esters, via a one-electron process.²⁶ The possibility of laccases being used in many biotechnological processes has been explored because oxygen is the final electron acceptor. These qualities have piqued the researcher’s interest in using laccases.

Laccase is typically a highly stable, industrially applicable, heat-resistant enzyme. This enzyme can eliminate xenobiotics and generate polymeric compounds employed in bioremediation procedures. Textile industry-produced phenols and dyes can be eliminated and detoxified using laccase.²⁷ Laccase is a promising method for treating organic micropollutants in wastewater. It effectively degrades and detoxifies various compounds, including pharmaceuticals, steroid hormones, personal care products, pesticides, and industrial chemicals. Laccases have been successfully utilized to remove pharmaceuticals, personal care products, biocides, endocrine-disrupting agents, steroid hormones, and microplastics. Although no commercially available laccase-based wastewater treatment preparations exist, studies demonstrate its effectiveness.²⁸

Agricultural wastes, including sawdust, banana peels, and rice bran, contain lignin and phenolic substances that increase laccase production.²⁹ There are numerous biotechnological uses for laccases in food, textile, paper, cosmetics, and other industries. This is because of their high oxidative capability. Laccases are also employed to break down agricultural items like pesticides and herbicides, protecting the environment from dangerous chemicals. Laccases remediate soil contaminated by oil hydrocarbons, treat wastewater containing textile industry dyes, and perform bio-bleaching procedures.^30,31 Laccases, known to have a huge substrate range, can oxidize a wide variety of aromatic chemicals, especially phenolic substrates.³² According to Guan et al.³³ the majority of laccases from different microorganisms are identified, described, and studied, especially the Streptomyces laccase from actinomycetes. Simulated textile effluents (STE) can be discolored by E. coli recombinant CotA laccase. Pure and unpurified CotA laccase decolorized dyes more rapidly when simulated textile effluents were buffered at pH 7.³⁴ In roughly four hours, the pure recombinant laccase eliminates over 93% of the tested colors at a basic pH of 9.0. Laccase-mediated dye degradation is an environmentally acceptable substitute for conventional dye degradation methods, which are sometimes costly and have negative effects.³⁵ Recombinant laccase, which is utilized in aquaculture wastewater bioremediation, is produced by B. vallismortis strain fmb103.³⁶ Five of the seven dyes that were tested-Evans Blue, Brom Cresol Purple, Remazol Brilliant Blue, Reactive Black 5, and Amido Black 10B-were destroyed by it.³⁷ Titanium dioxide, iron oxides, aluminum, mica flakes, cadmium, mercury, and lead are among the heavy metals known to be present in the many colored pigments used in the paint industry. According to reports, the laccase enzyme can break down heavy metals utilized in several industries. The laccase enzyme belongs to a broad class of enzymes known as polyphenol oxidases, which are helpful in the printing and tannery, baking, wine and beverage, textile, and pharmaceutical sectors. Laccase is one of the enzymes produced by microorganisms for bioremediation.

The purpose of this study is to separate bacteria from paint industry wastewater, screen for possible laccase-producing bacteria, and produce the laccase enzyme.³⁸

Laccases can be produced from filamentous fungi such as white rot fungi.³⁹ Strong Laccase producers include Pleurotus florida, P. ostreatus, P. pulmonarius,⁴⁰ P. tailandia.⁴¹ and Trichoderma.⁴² The fungi Trametes sp., Coriolopsis sp., Grifola sp., and many others also create laccases. The Rhus vernicifera tree served as the first source of laccase isolation.²² Bacteria such as E. coli, insects’ example Bombyx, Drosophila, Papilio, Schistocerca, etc., and plants that produce laccases include mango, peaches, and pines.³⁹

Dehydrogenase (EC1.1.1.1)
Dehydrogenases belong to the oxidoreductase family and are found in bacteria, yeast, plants, animals, & human beings. The bacteria’s cell-free extracts that break down xenobiotics manufactured industrially showed signs of polyethylene glycol dehydrogenase activity. Xenobiotic polyvinyl alcohol which is water soluble is broken down by recombinant polyvinyl alcohol dehydrogenase.^40,43 It has been discovered that aldehyde dehydrogenase is involved in the anabolic and catabolic processes of aromatic compounds and according to a protein expression analysis of Amycolatopsis tucumanensis DSM 45259 used in the biodegradation of phenanthrene, it is extensively and specifically expressed.⁴⁴ Table 1 lists a few dehydrogenases together with information about each one’s characteristics and function in bioremediation.

Lipases (EC 3.1.1.3)
Lipases are triacylglycerol ester hydrolyses that facilitate the conversion of triglycerides into glycerol and fatty acids. Lipases catalyze the reaction in which bonds are broken due to acid (acidolysis), alcohol (alcoholysis), and amino acids (aminolysis) in addition to hydrolysis. Lipases are the most adaptable biocatalysts. Plants, animals, and microbes all have lipases in varying concentrations. The most common source of lipases are microorganisms. Bacillus sp. is one of the most common bacteria that produces lipase. Bacillus alcalophilus, Staphylococcus caseolyticus, B. licheniformis, Serratia rubidaea, B. stearothermophilus, Pseudomonas aeruginosa, and Acinetobacter radioresistens are the most common producers of lipase.⁴⁵

Microbial lipases are used in industry to create biosensors, which are diagnostic instruments used to identify a range of illnesses. In addition to the pharmaceutical, polymerization, pulp and paper, and cosmetic industries, microbial lipases are commercially useful in the bioremediation of oil residues, petroleum pollutants, and effluents.^46,47 Lipases can accelerate the bioremediation of oily effluents that are generated from a range of sources that contain fats, proteins, and oils. Lipase from the bacteria Acinetobacter sp., Rhodococcus sp., and Mycobacterium sp., has been used to manage oil spills containing n-alkanes, aromatic hydrocarbons, and PAHs.^48,49

Pseudomonas aeruginosa lipase has been observed to degrade castor oil⁵⁰ and to remediate soil having waste oil released from industries. Bacteria that are isolated from soil polluted with motor oil produce lipase that helps in the remediation of hydrocarbon.⁵¹ In household laundry, lipases are used to reduce environmental contaminants and improve the effectiveness of detergent to get rid of stubborn grease or oil stains. Crude lipase isolated from Bacillus subtilis strain is used to lessen the impact of phosphate byproducts in laundry detergents.⁵² It was discovered that L. plantarum displayed the highest polycaprolactone (PCL) degradation efficiency in comparison with other lipases when it came to the breakdown of artificial polyester polycaprolactone (PCL) by co-cultures of L. brevis and L. plantarum lipases.⁵³ In fungus, lipase makers are Rhizopus (Rhizopus arrhizus, R. niveus), Penicillium, Aspergillus sp., etc. Lipases are expected to expand at the fastest rate among enzymes due to their extensive use in numerous industrial domains. Table 1 and 2 lists a few microbial lipases together with information about each one’s characteristics and bioremediation function.

Protease (E.C 3.4.21.12)
It is an enzyme that catalyzes the formation of peptide bonds in proteins and is a member of the hydrolase family. Proteases are mainly secluded from Bacillus and Aspergillus species. Microorganism-derived proteases are extremely important because of their low cost, high yield, and practical usage. They are widely used in industries like the food, leather, and wastewater treatment sectors.⁵⁴ Protease is used in the removal of polymers because it aids in the breakdown of -ester bonds created by polyhydroxyl butyrate (PHB) and c-linkages.⁵⁵ Because of the presence of keratin protein, which is insoluble in nature, animal horns, nails, poultry feces, and the shedding and molting of appendages are resistant to breakdown. Aside from their disagreeable smell, they are the cause of environmental pollution. By dissolving and recycling keratinous wastes into useful by-products, the protease enzyme keratinase aids in the breakdown of keratin proteins and can be applied to the bioremediation of bird excrement. Stenotrophomonas maltophilia KB13’s keratinase enzyme has shown its ability to use resistant keratinous waste in the biological deprivation of chicken feathers.⁵⁶ Protease producing bacteria has been discovered and identified from soil of tanneries.⁵⁷ These are used for silk degumming, leather, waste management, and in silver recovery.⁵⁸ Bacillus sp. FPF-1 has shown its ability to use resistant keratinous waste biomass from the agricultural industry (accession number MG214993) by degrading chicken feathers at an 82% rate.⁵⁹ By-products of feather degradation can be employed as ferric ion reducers, fertilisers for plant development, feed additives, free radical scavengers,⁶⁰ and hazardous hexavalent chromium-reducing agents. Keratinase is utilized in place of the conventional chemicals CaO and Na₂S in environmentally friendly enzymatic de-hairing operations in tanneries, which reduce pollution by preventing the release of harmful waste into water bodies.⁶¹ The bioremediation of marine crustacean wastes employed in the de-proteinization phase of chitin extraction uses protease enzymes. By producing alkaline protease, Bacillus licheniformis strain MP1 reduces the protein content of shrimp waste by 75%.⁶² At a temperature of 30 °C and a predominant pH of 7 to 8, Pseudomonas chlororaphis broke down the Impranil substrate while displaying protease activity (beta clearing zone) and esterase activity (alpha clearing zone).⁶³ By breaking down and transforming marine crustacean debris and keratinous waste products into beneficial molecules, proteases lessen environmental pollution.

Microorganisms are a natural source of commercial enzyme preparations as they be cultured in simple conditions, easy cell manipulation etc. About 40%-60% of all enzyme sales from microbial sources is of proteases.⁶⁴

Peroxidases (E.C. 1.11.1.7)
They are oxidoreductases that utilizes free radical mechanism for transforming a variety of chemical substances into oxidized or polymerized products.⁶⁵ Peroxidase activity transforms ferricyanides and ascorbic acid into innocuous components. They lessen water pollution by helping in the bioremediation of wastewater contaminated with chlorinated phenolic chemicals, cresol, and phenol. Peroxidases are suitable enzymes that help in the development of enzyme-linked immunosorbent assay (ELISA) kits, as they can produce chromogenic products at very low concentrations.⁶⁶ These enzymes are used in several industrial and analytical bioprocesses because of their strong reduction potential. They degrade artificial dyes such as brilliant blue, azo dyes, Cibacron red, remazol blue etc.^67,68 Peroxidases are also used in the paper industry to hydrolyze cellulose and lignin into carbon dioxide and water, respectively, which breaks down wood components.

Microbes, plants, and animals are the main sources of peroxidases. There have been reports of peroxidase production from a variety of plant sources, including papaya (Carica papaya), banana (Musa paradisiaca), horseradish (Armoracia rusticana),⁶⁹ and so on. Pseudomonas sp., Escherichia coli, and Bacillus sp. are the main bacterial strains for production of peroxidase. However, Pleurotus ostreatus., Umbelopsis isabelline, Auricularia sp., and, Thanatephorus sp. have been reported to be efficient peroxidase producers in the case of fungal strains.^70,71

Cellulases (E.C. 3.2.1.4)
The hydrolysis of cellulosic substrates into monomeric products is facilitated by the cellulase enzymes. They are created when microbial strains hydrolyze the α-1,4-glycosidic linkages of cellulose while growing on cellulosic substrates. Several strains of bacteria, yeast, and fungi have been found to contain cellulases. Because they can use additional pathways to develop higher cellulase activity, fungi are the primary microbiological species that manufacture cellulase enzymes. Aspergillus and Trichoderma are well-known fungal genera that produce cellulase. Strong cellulase production has been reported for A. niger, T. asperellum,⁷² Trichoderma viride,⁷³ A. fumigatus,⁷⁴ A. ellipticus,⁷⁵ and Aspergillus protuberus. Myceliophthora thermophile, Penicillium echinulatum, and Rhizopus oryzae are among the other fungal strains that may have cellulase activity. Thermomonospora sp., Cellulomonas sp., Microbispora sp., Clostridium sp., Cellvibrio sp., and Ruminococcus sp. are among the bacteria that are known to be powerful cellulose-producing genera.⁷⁶

Cellulase enzyme is mostly utilized in textile industry for bio-polishing, to modify the structure of fibres and are also used in the last phase of textile manufacturing to soften and lessen the tendency of fabric to pill as well as to remove the starch sizing.⁷⁷

Microbial enzymes mechanism for bioremediation
In nature, several genes are known to express different types of enzymes and that regulate how these enzymes function and what kind of structure they should adopt to perform a certain function. The numerous protein folds of the enzyme are regulated by highly distinct sets of genes, which not only adapt the structure of the enzyme for a variety of uses but also determine the catalytic mechanism, which establishes the function of enzymes at a given site. Figure 3 shows the mechanism of action for some of the most important microbial enzymes utilized in bioremediation.

Free radicals are created when laccase oxidizes the substrate. Laccase-mediated catalysis can be expanded by utilizing mediators. Laccase oxidizes mediators, which are organic molecules with a low molecular weight. The glycerol backbone of a lipid substrate serves as the typical site of lipase activity in the case of the lipase enzyme.

Electrons are transferred from the substrate to an electron carrier by dehydrogenase enzyme. NAD⁺, FAD, and NADP⁺ are common electron acceptors utilized by this subclass. In this process, electron carriers are reduced and are regarded as oxidizers of the substrate. Co-enzymes such as flavin groups, nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN), or nicotinamide adenine dinucleotide phosphate (NADP) catalyze the reaction with dehydrogenase enzymes.⁸³ The dehydrogenase enzyme, which is present in all living organisms, transports hydrogen atoms from organic transporters to electron-acceptor substances.⁸⁴ Hydroxylase enzymes have active sites containing metal that can catalyze the reactions.

Dioxygen molecules can serve as an electron acceptor for oxidases to use in catalyzing processes.⁸³ Oxidases transfer electrons using a variety of substances, including metals and cofactors. These substances consist of amine oxidases, metals based on alcohol or flavin, or both.⁸⁵ Utilizing molecular oxygen and NADH or NADPH as a cofactor, cytochrome P450 produces oxidized products and carbon substrate.²⁴ As a source of electrons, cytochrome P450 uses ferredoxin reductase and ferredoxin for catalytic function.

Enzymatic biodegradation potential for sustainability
The enzymatic activity of microorganisms that convert harmful substances into less toxic or non-hazardous compounds is the primary focus of biological remediation of toxic contaminants.^96,97 Using microbial enzymes over microbial cell majorly depends on the Standardizable activity, ease of handling and storage, more selectivity, improved mobility because of their smaller size, ability to function in the face of high concentrations of harmful substances, and biodegradability.⁹⁸ Microbial enzymes enhance the remediation process by yielding non-flammable, non-corrosive byproducts that can be easily disposed of Perpetuini et al.⁹⁹ It has been seen that immobilization increases the stability and lifetime of enzymes by reducing enzymatic activity.^100,101 Recombinant DNA technology has helped to generate more effective enzymes in significant amounts.¹⁰² Because enzymes are sustainable, naturally occurring catalysts derived from renewable resources, microbially produced enzyme remediation is a safe and cost-effective bioremediation method. They use as many natural products as possible and are biodegradable.¹⁰³ Enzyme-mediated degradation of toxic pollutants provides an environmentally friendly technique by reducing post-treatment environmental hazards, making it more socially acceptable.⁵⁴

Recombinant microbial enzymes produced by different expression systems
Studies have demonstrated effective methods to create various microbial enzymes by diverse microorganisms (Table 3) using a variety of study approaches that have been improved in recent years. Recombinant laccase has been investigated for the treatment of dyes, phenolic compounds, insecticides, and polycyclic aromatic hydrocarbons due to its capacity to oxidize a variety of substrates.¹⁰⁴ Laccase-mediated degradation has been investigated for the treatment of a number of pollutants, including dyes, phenolic compounds, insecticides, and polycyclic aromatic hydrocarbons. Endo-1,4-glucanase-encoding Aspergillus fumigatus gene (Afu6g01800) was cloned in the pET-28a (+) vector and was expressed in the Rosetta TM (DE3) strain of E. coli. The findings of this study demonstrated that the afegl7 enzyme belongs to the GH7 family. The afegl7 gene encodes a protein of 460 amino acids, a CBM1 domain at residues 424-460, and a molecular weight of 52kDa.¹⁰⁵

Table (3):
Recombinant DNA technology produced microbial enzymes for enhanced bioremediation

Laccase cotA produced from Bacillus subtilis, was expressed in E. coli DH5 using the pMD18-T vector, is well known for its synthetic dye efficiency.³⁰ Another laccase, CueO, derived from E. coli K12, was expressed in Pichia pastoris GS115 using the pHBM905BDM vector, demonstrating efficacy in the textile effluents dye decolorization. Laccase Fmb-rL103, from similarly Bacillus vallismortis fmb-103 and expressed in E. coli BL21 using the pMD19-Tlac103 vector, is effective against triphenyl methane dyes. Cellobiohydrolase CBH1 from Aspergillus niger, expressed in Pichia pastoris, is effective in the degradation of pulp and cellulose. Similarly, endoglucanase ReEG I, derived from Trichoderma reesei and expressed in Pichia shepherds, can degrade various substrates including pulp, cellulose, oat xylan, birch xylan, and corn straw.

Cytochrome P105D1, sourced from Streptomyces griseus and expressed in Acinetobacter calcoaceticus using the pSP19g10L vector, plays a key role in the breakdown of various pollutants and herbicides.¹⁰⁸ It has been observed that the Dehydrogenase from Rhodococcus sp. P14, expressed in E. coli with pET-32a, significantly contributes to the bioremediation of steroids.¹⁰⁶ A study suggests that dehalogenase produced by Ochrobactrum species, and expressed in E. coli BL21 (DE3) using the pET30a-a6 vector, can be used for the degradation of the environmental contaminant Tetra Bromo Bis Phenol A (TBBPA).¹¹⁰ Thus, Several recombinant enzymes have been developed and expressed in various host systems for applications in biodegradation and bioremediation.